Cy5 total protein normalization in Western blot analysis
Cy5 total protein normalization in Western blot analysis
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DOI：
10.1016/j.ab.2015.06.017
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发表时间：
2015-10-01
影响因子：
2.9
通讯作者：
Ronn, Ola
中科院分区：
文献类型：
--
作者：
Hagner-McWhirter, Asa;Laurin, Ylva;Ronn, Ola
蛋白质印迹是一种广泛使用的方法，用于分析复杂蛋白质样品中的特定靶蛋白。管家蛋白通常用于标准化，以纠正不均匀的样品负载，但这些需要仔细验证，因为表达水平可能随细胞类型和处理而变化。我们提出了一种新的、更可靠的标准化方法，使用 Cy5 预标记的总蛋白作为上样对照。我们使用基于 Cy5 N-羟基琥珀酰亚胺酯标记的预标记方案，可产生线性信号响应。在 2.5 至 20.0 μg 中国仓鼠卵巢细胞裂解物蛋白的整个加载范围内，我们获得了细胞外信号调节激酶 (ERK1/2) 靶标与 Cy5 总蛋白控制信号的比率之间的 7% 的低变异系数 (CV)。使用肌动蛋白或微管蛋白作为标准化对照的相应实验导致 CV 分别为 13% 和 18%。 3-磷酸甘油醛脱氢酶不产生比例信号，不适合在这些细胞中标准化。标记和未标记样品的 ERK1/2 信号比较表明 Cy5 预标记不影响抗体结合。通过使用总蛋白归一化，我们以高置信度分析了 PP2A 和 Smad2/3 水平。 (C) 2015 Elsevier Inc. 保留所有权利。
Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0 mu g of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. (C) 2015 Elsevier Inc. All rights reserved.